Georgy Bakalkin

NF-κB-like factors in the murine brain. Developmentally-regulated and tissue-specific expression

Abstract NF-κB and related factors are important transducers of external signals to the cell nucleus. They are abundant in the brain, where they may be significant for the regulation of gene transcription in plasticity-related processes for instance, via activation of protein kinase C. The subunit composition and levels of these factors in the mouse and rat brain and other tissues, using an assay based on gel retardation of the oligonucleotides corresponding to the κB DNA-element, are reported here. Three major κB-binding factors were observed. Factors I and II were activated by the dissociating agent deoxycholate. DNA protein cross-linking and antibody neutralization experiments suggest that factor I is a heterodimer of c-Rel and p65; factor II is a heterodimer of p50 and p65 (authentic NF-κB), and of p50 and c-Rel; factor III is the p50 homodimer (KBF1). All three factors were generally expressed in the 17-day-old rat embryo and 5-day-old pup, whereas in the adult rat, expression was more limited and showed certain tissue specificity. Factor II was the most generally expressed and the only factor observed in adult brain. Factor I was only detected in the adult testis whereas factor III was observed in the adult spleen and, in small amounts, in the liver and lung. Two minor κB-specific factors (A and B), distinctive to the brain and spleen, respectively, showed very slow gel mobility. Their estimated molecular weights were about 125 kDa and 95 kDa, respectively. Expression of factor A was stable in the rat brain during development. Factor A may be identical to a previously described brain-specific factor, BETA (Korner et al., Neuron , 3 (1989) 563–572). Thus, the expression pattern of κB-binding activities is apparently developmentally regulated and tissue-specific particularly in the adult. In the adult mouse and rat brain, only factors II (probably NF-κB and p50/c-Rel heterodimer) and A (probably BETA) could be observed.

Study of molecular events in cells by fluorescence correlation spectroscopy

Abstract.To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

PATHOBIOLOGY OF DYNORPHINS IN TRAUMA AND DISEASE

Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.

Neuroadaptations in human chronic alcoholics : dysregulation of the NF-κB system

Background Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-κB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. Methods and Findings Analysis of DNA-binding of NF-κB (p65/p50 heterodimer) and the p50 homodimer as well as NF-κB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant κB binding factor in analyzed tissues. NF-κB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-κB target DNA sites, κB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with κB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. Conclusions We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-κB, when repeated over years downregulate RELA expression and NF-κB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of κB regulated genes. Alterations in expression of p50 homodimer/NF-κB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.

Validation of endogenous controls for quantitative gene expression analysis : Application on brain cortices of human chronic alcoholics

Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.

Big dynorphin as a putative endogenous ligand for the κ‐opioid receptor

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphinu2003A (Dynu2003A), and dynorphinu2003B (Dynu2003B) are endogenous opioid peptides that activate the κ‐opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32‐amino acid prodynorphin‐derived peptide consisting of Dynu2003A and Dyn B, with human KOR, μ‐ (hMOR) and δ‐ (hDOR) opioid receptors and opioid receptor‐like receptoru20031 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dynu2003A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dynu2003B. Dynu2003A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dynu2003B demonstrated low selectivity. In contrast, Big Dyn activated Gu2003proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR‐mediated activation of Gu2003proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of Gu2003proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dynu2003B being approximately 1u2003:u20033. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR‐mediated activation of Gu2003proteins.

Prodynorphin storage and processing in axon terminals and dendrites

The classical view postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans‐Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN‐containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and striatum, PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium‐induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission. —Yakovleva, T., Bazov, I., Cebers, G., Marinova, Z., Hara, H., Ahmed, A., Vlaskovska, M., Johansson, B., Hochgeschwender, U., Singh, I. N., Bruce‐Keller, A. J., Hurd, Y. L., Kaneko, T., Terenius, L., Ekström, T. J., Hauser, K. F., Pickel, V. M., Bakalkin, G. Prodynorphin storage and processing in axon terminals and dendrites FASEB J. 20, E1430 –E1440 (2006)

Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism.

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.

Prodynorphin knockout mice demonstrate diminished age-associated impairment in spatial water maze performance.

Dynorphins, endogenous kappa-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin (Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13-17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3-6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

Dysregulation of dynorphins in Alzheimer disease

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.